Methods of expressing gram-negative glycosaminoglycan synthase genes in gram-positive hosts

ABSTRACT

The present invention relates to a Gram-negative glycosaminoglycan gene and methods of making and using same. The present invention relates to recombinant Gram-positive host cells containing a Gram-negative glycosaminoglycan synthase gene, and methods of producing glycosaminoglycans using such recombinant host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of U.S. Ser. No.60/765,140, filed Feb. 2, 2006. This application is acontinuation-in-part of U.S. Ser. No. 11/042,530, filed Jan. 24, 2005;which is a continuation of U.S. Ser. No. 09/842,484 filed Apr. 25, 2001,now abandoned, which claims benefit under 35 U.S.C. 119(e) ofprovisional application U.S. Ser. No. 60/199,538, filed Apr. 25, 2000.Said U.S. Ser. No. 09/842,484 is also a continuation-in-part of U.S.Ser. No. 09/283,402, filed Apr. 4, 1999, now abandoned; and said U.S.Ser. No. 09/842,484 is also a continuation-in-part of U.S. Ser. No.09/437,277, filed Nov. 10, 1999, now U.S. Pat. No. 6,444,447, issuedSep. 3, 2002. The contents of each of the above-referenced patents andpatent applications are hereby expressly incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The government owns certain rights in the present invention pursuant toa grant from the National Institutes of Health (GM56497) and a grantfrom the National Science Foundation (MCB-9876193).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a chondroitin synthase gene and methodsof making and using same. In more particular, but not by way oflimitation, the present invention relates to a chondroitin synthase genefrom Pasteurella multocida and methods of using same in a gram-positivehost.

2. Background Information Relating to the Invention

Glycosaminoglycans [GAGs] are long linear polysaccharides consisting ofdisaccharide repeats that contain an amino sugar and are found in mostanimals. Chondroitin [β(1, 4)GlcUA-β(1,3)GalNAc]_(n), heparin/heparan[α(1,4)GlcUA-β(1, 4)GlcNAc]_(n), and hyaluronan [β(1, 4)GlcUA-β(1,3)GlcNAc]_(n) are the three most prevalent GAGs found in humans.Chondroitin and heparin in animals typically have n=20 to 100, whilehyaluronan typically has n=10³. Chondroitin and heparin are synthesizedas glycoproteins and are sulfated at various positions in vertebrates.Hyaluronan is not sulfated in vertebrates. A substantial fraction of theGlcUA residues of heparin and chondroitin are epimerized to formiduronic acid.

Many lower animals possess these same GAGs or very similar molecules.GAGs play both structural and recognition roles on the cell surface andin the extracellular matrix. By virtue of their physicalcharacteristics, namely a high negative charge density and a multitudeof polar hydroxyl groups, GAGs help hydrate and expand tissues. Numerousproteins bind selectively to one or more of the GAGs. Thus the proteinsfound on cell surfaces or the associated extracellular matrices (e.g.,CD44, collagen, fibronectin) of different cell types may adhere orinteract via a GAG intermediate. Also GAGs may sequester or bind certainproteins (e.g. growth or coagulation factors) to cell surfaces.

Certain pathogenic bacteria produce an extracellular polysaccharidecoating, called a capsule, which serves as a virulence factor. In a fewcases, the capsule is composed of GAG or GAG-like polymers. As themicrobial polysaccharide is identical or very similar to the host GAG,the antibody response is either very limited or non-existent. Thecapsule is thought to assist in the evasion of host defenses such asphagocytosis and complement. Examples of this clever strategy ofmolecular camouflage are the production of an authentic HApolysaccharide by Gram-negative Type A Pasteurella multocida andGram-positive Group A and C Streptococcus. The HA capsule of thesemicrobes increases virulence by 10₂ to 10³-fold as measured by LD₅₀values, the number of colony forming units that will kill 50% of thetest animals after bacterial challenge.

The invasiveness and pathogenicity of certain E. coli strains has alsobeen attributed to their polysaccharide capsules. Two Escherichia colicapsular types, K4 and K5, make polymers composed of GAG-like polymers.The E. coli K4 polymer is an unsulfated chondroitin backbone decoratedwith fructose side-branches on the C3 position of the GIcUA residues.The E. coli K5 capsular material is a polysaccharide, called heparosan,identical to mammalian heparin except that the bacterial polymer isunsulfated and there is no epimerization of GlcUA to iduronic acid.

The studies of GAG biosynthesis have been instrumental in understandingpolysaccharide production in general. The HA synthases were the firstGAG glycosyltransferases to be identified at the molecular level. Theseenzymes utilize UDP-sugar nucleotide substrates to produce largepolymers containing thousands of disaccharide repeats. The genesencoding bacterial, vertebrate, and viral HAS enzymes have been cloned.In all these cases, expression studies demonstrated that transformationwith DNA encoding a single HAS polypeptide conferred the ability offoreign hosts to synthesize HA. Except for the most recent HAS to beidentified, P. multocida PmHAS, these proteins have similar amino acidsequences and predicted topology in the membrane. Two classes of HASshave been proposed to exist based on these structural differences aswell as potential differences in reaction mechanism

The biochemical study of chondroitin biosynthesis in vertebrates wasinitiated in the 1960s. Only recently have the mammalian enzymes forelongation of the polysaccharide backbone of chondroitin beententatively identified by biochemical means. An 80-kDa GlcUA transferasefound in vertebrate cartilage and liver was implicated in thebiosynthesis of the chondroitin backbone by photoaffinity labeling withan azidoUDP-GlcUA probe. A preparation from bovine serum with theappropriate GalNAc- and GlcUA-transferase activities in vitro wasobtained by conventional chromatography, but several bands on SDSpolyacrylamide gels (including a few migrating ˜80 kDa) were observed.Several genes called ChSy have been recently identified that encode theenzymes that polymerize the chondroitin backbone in animals and humans;these proteins are not homologous to the Pasteurella PmCS gene describedherein.

Chondroitin polysaccharide ([β(1,3)GalNAc-β(1,4)GlcUA]_(n); wheren=˜10-2000) has use as a hyaluronan (HA) polysaccharide substitute inmedical or cosmetic applications. Both chondroitin and hyaluronan formviscoelastic gels (suitable for eye or joint applications) orhydrophilic, hygroscopic materials (suitable for moisturizer or wounddressings). Unmodified or underivatized chondroitin is not known toexist or, if present, in very small quantities in the human body. Themain advantage is that byproducts of natural HA degradation (by shear,enzyme, radical or oxidation processes) have certain biologicalactivities with respect to vascularization, angiogenesis, cancer, tissuemodulation, but similar byproducts of chondroitin (in the proposedunsulfated, unmodified state) may not have the same biological activity.The chondroitin polymers are more inert, loosely speaking, than theanalogous HA molecule. Chondroitin from either P. multocida Type F or arecombinant host containing the Pasteurella-derived or Pasteurella-likesynthase gene will serve as an alternative biomaterial with uniqueproperties.

With respect to related microbial GAG synthases other than the HASs, theE. coli K5 glycosyltransferases that synthesize heparosan have beenidentified by genetic and biochemical means. In contrast to the HASs, itappears that two proteins, KfiA and KfiC, are required to transfer thesugars of the disaccharide repeat to the growing polymer chain. Thechondroitin-backbone synthesizing enzyme of E. coli K4 has beenenzymatically characterized, and the gene encoding the relevantglycosyltransferases, KfoC, was recently discovered; it is veryhomologous (e.g., DNA will cross-hybridize) to the PmCS gene describedherein. The KfoC enzyme performs the same reaction as PmCS in vitro, butthe former protein appears less robust.

Many P. multocida isolates produce GAG or GAG-like molecules as assessedby enzymatic degradation and removal of the capsule of living bacterialcells. Type A P. multocida, the major fowl cholera pathogen, makes acapsule that is sensitive to hyaluronidase. Subsequent NMR structuralstudies of capsular extracts confirmed that HA was the majorpolysaccharide present. A specific HA-binding protein, aggrecan, alsointeracts with HA from Type A P. multocida. Two other distinct P.multocida types, a swine pathogen, Type D, and a minor fowl cholerapathogen, Type F, produce polymers that are chondroitin orchondroitin-like based on the observation that their capsules aredegraded by Flavobacterium chondroitin AC lyase. After enzymatic removalof the capsule, both types were more readily phagocytosed by neutrophilsin vitro. The capsule of Type D cells, but not Type F cells, is alsoreported to be degraded by heparinase III, suggesting a heparin-typemolecule is present, too.

Parent application U.S. Ser. No. 09/842,484 discloses the identificationof PmCS (P. multocida Chondroitin Synthase), the first chondroitinsynthase to be identified and molecularly cloned from any source.Interestingly, a single polypeptide is responsible for thecopolymerization of the GlcUA and GalNAc sugars, and thus PmCS is asingle protein that is a dual-action transferase that catalyzes thepolymerization of UDP-GlcUA and UDP-GalNAc to form chondroitin. The '484parent application also identified the Type F capsular polymer as anunsulfated chondroitin polymer, and identified organisms with thechondroitin synthase gene (Type F P. multocida) as new sources ofunsulfated chondroitin polymer.

Certain glycosaminoglycan synthase enzymes (namely enzymes fromStreptococcus, virus and vertebrates) appear to be self-contained,meaning that they possess both (a) sugar addition or polymerizationactivity (glycosyltransferase) as well as (b) sugar export functions(transport the polymer across the membrane to the outside of the cellwhere it can be easily harvested). Certain other distinct enzymes,including those from Pasteurella, can catalyze glycosaminoglycanpolymerization, but in general are thought to not be able to completethe sugar export step without assistance of other proteins.Gram-negative bacteria have two membranes and are generally thought toneed more transport machinery than Gram-positive bacteria with a singlemembrane. In addition, Pasteurella (as well as other sugar-producingGram-negative pathogens such as E. coli) is not a preferred host for theproduction of glycosaminoglycans intended for use in animals or humansdue to the presence of endotoxins or lipopolysaccharides (moleculesderived from their outer membrane due to the possibility of inducingshock, etc.

The Gram-negative bacteria capable of GAG biosynthesis, Escherichia coliand Pasteurella multocida, possess two lipid membranes, and theircapsule loci encode many transport-associated proteins in addition tothe glycosyltransferases and the UDP-GlcUA forming enzymes (˜10-18kilobases; Roberts, 1996; Townsend et al, 2001). Although many detailsare not well understood, in the best-studied model, the E. coli Group IIcapsular system, it appears that transport of the nascent polymer chainrequires an apparatus composed of at least 7 distinct polypeptidespecies (Whitfield and Roberts, 1999; Silver et al, 2001). Briefly, acomplex containing KpsC,M,S,T assembles on the inner membrane andinteracts with the KfiA,B,C catalytic complex. KpsM and T form theATP-binding cassette (ABC) transporter. A periplasmic protein, KpsD, anda dimer of another inner membrane protein, KpsE, help transport thepolymer across the periplasmic space (Arrecubieta, 2001). A porincomplex in the outer membrane is recruited to transport the growingpolysaccharide chain out of the cell. Certain Kps mutants polymerize thecapsular polysaccharide chain, but possess faulty translocationresulting in polymer accumulation in the cytoplasm or periplasm. P.multocida is also thought to have a Group II-like transport system basedon the sequence similarities and gene arrangement of its putativetransport proteins to the E. coli proteins.

In the case of PmHAS and PmCS, the carboxyl-terminal tail was thought tocontain a docking segment that interacts with the transport mechanism(Jing and DeAngelis, 2000). However, the region(s) of E. coli K5 enzymesresponsible for docking to the transport apparatus is not known andthere is no obviously similar sequence to the carboxyl-terminus of thePasteurella enzymes. Polymer transport across membranes is a difficultphenomenon to study. Therefore, it is thought that any recombinantmicrobial system endeavoring to utilize the Pasteurellaglycosaminoglycan synthases must solve or circumvent the transportproblem.

Therefore, there is a need felt in the art to provide methods ofexpressing Gram-negative glycosaminoglycan synthase gene in aGram-positive bacterial host background to produce GAGs by fermentationin vivo. It is to such methods of expressing a Gram-negativeglycosaminoglycan synthase gene in a Gram-positive host that the presentinvention is directed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates schematics of plasmid Maps utilized in a ChondroitinSynthase Production System for Bacillus. Top panel: A cassette with aBacillus ribosome binding site (RBS) and chondroitin synthase gene(PmCS) was cloned into pHCMC04 (pro=xylA Xylose-inducible promoter;con=xylR) and into pHCMC05 (pro=Pspac promoter/IPTG inducible; con=Lacl) via the BamHI and SmaI sites of the multiple cloning site. Thesevectors will replicate in and drug-selected in both E. coli (ampicillinwith bla) and B. subtilis (chloroamphenicol with cat). Bottom panel: Ina similar fashion, a cassette with a Bacillus ribosome bindingsite-chondroitin synthase gene (PmCS)-Bacillus ribosome bindingsite-Bacillus UDP-GalNAc/UDP-GlcNAc epimerase gene (GalE) was clonedinto pHCMC04 (pro=xylA Xylose-inducible promoter; con=xylR) and intopHCMC05 (pro=Pspac promoter/IPTG inducible; con=Lacl) via the BamHI andSmaI sites of the multiple cloning site.

FIG. 2 illustrates an agarose gel analysis of recombinantBacillus-derived Chondroitin. Samples from cultures of Bacillus subtiliswith vector alone control plasmid (lane N) or containing the PmCScassette plasmid (lane 1) or the PmCS-GalE cassette plasmid (lanes 2, 3)were run on an agarose gel and stained with StainsAll. The strains withPmCS produce a chondroitin polymer at ˜100-300 kDa (based on HAstandards, lane S) that is not found in the “no insert” control strain.

FIG. 3 illustrates agarose gel analysis of recombinant Bacillus-derivedChondroitin and the effect of hyaluronidase challenge. Samples fromcultures of Bacillus subtilis with either a vector alone control plasmid(lane N) or the PmCS-GalE cassette plasmid (lane 4) were prepared. Onehalf of the sample was treated with testicular hyaluronidase (HAase), anenzyme known to cleave both HA and chondroitin polymer to very smallchains; the other half of the sample was saved. The HAase treated (+)and untreated (−) samples were then run on an agarose gel and stainedwith StainsAll. The strains with PmCS gene produce a chondroitin polymerat ˜100-300 kDa (based on HA standards, lane S) that is not found in the“no insert” control strain. The polymer is sensitive to enzymaticdegradation as is authentic chondroitin.

FIG. 4 illustrates Fluorophore-Assisted Carbohydrate Electrophoresis(FACE) gel analysis of recombinant Bacillus-derived Chondroitin. Asample from a culture of Bacillus subtilis with the PmCS-GalE cassettein the pHCMC05 plasmid (lane B) as well as authentic HA (lane HA) orauthentic Chondroitin (lane C) were prepared for FACE by chondroitinasedigestion and fluor-tagging of the resulting disaccharides. The sampleswere then run on a polyacrylamide gel and imaged with UV light. Mixingthe recombinant Bacillus sample with the authentic chondroitin sample(lane B+C) shows only a single band corresponding to a chondroitindisaccharide (delta-GlcUA-GalNAc) that proves that the strain with PmCSgene produces the authentic chondroitin.

FIG. 5 illustrates a Southern blot showing the hybridization of achondroitin synthase probe with either Pasteurella (denoted as P) or E.coli K4 (denoted as K) DNA. The ethidium bromide stained gel shows thetotal DNA (L is the DNA ladder). Thus, sequences encoding otherchondroitin synthase/glycosyltransferases genes similar to PmCS can beidentified in other microbial species.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al.Current Protocols in Immunology (Current Protocols, Wiley Interscience(1994)), which are incorporated herein by reference. The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

All patents, patent applications, publications, and literaturereferences cited in this specification are hereby expressly incorporatedherein by reference in their entirety.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “nucleic acid segment” and “DNA segment” areused interchangeably and refer to a DNA molecule which has been isolatedfree of total genomic DNA of a particular species. Therefore, a“purified” DNA or nucleic acid segment as used herein, refers to a DNAsegment which contains a Chondroitin Synthase (“CS”) coding sequence yetis isolated away from, or purified free from, unrelated genomic DNA, forexample, total Pasteurella multocida or, for example, mammalian hostgenomic DNA. Included within the term “DNA segment”, are DNA segmentsand smaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified pmCS(Pasteurella multocida Chondroitin Synthase) gene refers to a DNAsegment including Chondroitin Synthase coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case pmCS, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or DNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to, or intentionally left in the segment by the hand of man.

Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe CS gene from Pasteurella multocida. One such advantage is that,typically, eukaryotic enzymes may require significant post-translationalmodifications that can only be achieved in a eukaryotic host. This willtend to limit the applicability of any eukaryotic CS gene that isobtained. Moreover, those of ordinary skill in the art will likelyrealize additional advantages in terms of time and ease of geneticmanipulation where a prokaryotic enzyme gene is sought to be employed.These additional advantages include (a) the ease of isolation of aprokaryotic gene because of the relatively small size of the genome and,therefore, the reduced amount of screening of the corresponding genomiclibrary and (b) the ease of manipulation because the overall size of thecoding region of a prokaryotic gene is significantly smaller due to theabsence of introns. Furthermore, if the product of the ChondroitinSynthase gene (i.e., the enzyme) requires posttranslationalmodifications or cofactors, these would best be achieved in a similarprokaryotic cellular environment (host) from which the gene was derived.

The term “Gram-positive host cell” as used herein will be understood torefer to prokaryotic cells recognized by a Gram staining procedure asGram-positive. Gram-positive organisms have a cell wall that isrelatively thick (approximately 15-80 nm across), and consists of anetwork of peptidoglycan; this allows the cell wall to retain the basicdye utilized in the Gram stain. Examples of Gram-positive host cellsthat may be utilized in accordance with the present invention include,but are not limited to, Bacillus, Staphylococcus, Peptococcus,Lactobacillus, Lactococcus, Actinomyces, and Streptomyces and theirallies.

In one embodiment, the Gram-positive host cell is a Bacillus cell, suchas but not limited to, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus metaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis and Bacillusthuringienisis cells.

Preferably, DNA sequences in accordance with the present invention willfurther include genetic control regions which allow the expression ofthe sequence in a selected recombinant host. Of course, the nature ofthe control region employed will generally vary depending on theparticular use (e.g., cloning host) envisioned.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode aChondroitin Synthase gene such as but not limited to pmCS. In the caseof pmCS, the isolated DNA segments and recombinant vectors incorporatingDNA sequences which include within their amino acid sequences an aminoacid sequence in accordance with SEQ ID NO:2. Moreover, in otherparticular embodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of anChondroitin Synthase gene or DNA, and in particular to an ChondroitinSynthase gene or cDNA, corresponding to Pasteurella multocidaChondroitin Synthase—pmCS. For example, where the DNA segment or vectorencodes a full length Chondroitin Synthase protein, or is intended foruse in expressing the Chondroitin Synthase protein, particularnon-limiting examples of sequences are those which are essentially asset forth in SEQ ID NO:1 or 2.

Amino acid segments having chondroitin synthase activity may be isolatedby the methods described herein. The term “a sequence essentially as setforth in SEQ ID NO:2” means that the sequence substantially correspondsto a portion of SEQ ID NO:2 and has relatively few amino acids which arenot identical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:2. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein, as agene encoding a sequence essentially as set forth in SEQ ID NO:2, andthat is associated with the ability of prokaryotes to producechondroitin or a “chondroitin like” polymer or a chondroitin synthasepolypeptide.

One of ordinary skill in the art would appreciate that a nucleic acidsegment encoding enzymatically active chondroitin synthase may containconservative or semi-conservative substitutions to the sequences setforth in SEQ ID NOS:1 and/or 2 and yet still be within the scope of theinvention. For example, a biologically functional equivalent may be anamino acid sequence comprising SEQ ID NO:2 with 0 to 20 conservativeamino acid substitutions. Alternatively, a biologically functionalequivalent may be an amino acid sequence comprising SEQ ID NO:2 with 0to 10 conservative amino acid substitutions. As shown in the parentapplication (U.S. Ser. No. 11/042,530) and in Jing and DeAngelis(Glycobiology, 13:661-671 (2003)), the catalytic residues of PmCS arefound in the amino acid residues of the central portion of the openreading frame; thus, truncation or major substitutions of the openreading frame at either termini is also possible while retaining sugartransfer activity.

In particular, the art is replete with examples of practitioner'sability to make structural changes to a nucleic acid segment (i.e.,encoding conserved or semi-conserved amino acid substitutions) and stillpreserve its enzymatic or functional activity. See for example: (1)Risler et al. “Amino Acid Substitutions in Structurally RelatedProteins. A Pattern Recognition Approach” J. Mol. Biol. 204:1019-1029(1988) [“ . . . according to the observed exchangeability of amino acidside chains, only four groups could be delineated; (i) Ile and Val; (ii)Leu and Met, (iii) Lys, Arg, and Gin, and (iv) Tyr and Phe.”]; (2)Niefind et al. “Amino Acid Similarity Coefficients for Protein Modelingand Sequence Alignment Derived from Main-Chain Folding Anoles.” J. Mol.Biol. 219:481-497 (1991) [similarity parameters allow amino acidsubstitutions to be designed]; and (3) Overington et al.“Environment-Specific Amino Acid Substitution Tables: Tertiary Templatesand Prediction of Protein Folds,” Protein Science 1:216-226 (1992)[“Analysis of the pattern of observed substitutions as a function oflocal environment shows that there are distinct patterns . . . ”Compatible changes can be made.]

These references and countless others available to one of ordinary skillin the art, indicate that given a nucleic acid sequence, one of ordinaryskill in the art could make substitutions and changes to the nucleicacid sequence without changing its functionality. Also, a substitutednucleic acid segment may be highly identical and retain its enzymaticactivity with regard to its unadulterated parent, and yet still fail tohybridize thereto.

One of ordinary skill in the art would also appreciate thatsubstitutions can be made to the pmCS nucleic acid segment listed in SEQID NO:1 without deviating outside the scope and claims of the presentinvention. Standardized and accepted functionally equivalent amino acidsubstitutions are presented in Table I. TABLE I Conservative and Semi-Amino Acid Group Conservative Substitutions NonPolar R Groups Alanine,Valine, Leucine, Isoleucine, Proline, Methionine, Phenylalanine,Tryptophan Polar, but uncharged, Glycine, Serine, Threonine, Cysteine, RGroups Asparagine, Glutamine Negatively Charged Aspartic Acid, GlutamicAcid R Groups Positively Charged Lysine, Arginine, Histidine R Groups

The present invention is not limited to the specific chondroitinsynthase nucleotide and amino acid sequences disclosed herein. Rather,the present invention also includes chondroitin synthase nucleotide andamino acid sequences from other genus and species that can be identifiedutilizing the sequences and methods described herein. For example, theuse of PmCS sequences described herein allowed for the identification ofa homolog, E. coli K4 kfoC, by hybridization as described in detailherein after. Such identified sequences also fall within the scope ofthe present invention, and may be defined in terms of sequence identityto the PmCS sequences or the ability to hybridize to a complement of thepmCS sequences disclosed herein, as described in detail herein after.

Another preferred embodiment of the present invention is a recombinantvector containing (i) a purified nucleic acid segment in accordance withSEQ ID NO:1, or (ii) a purified nucleic acid segment that encodes aprotein in accordance with SEQ ID NO:2. As used herein, the term“recombinant vector” refers to a vector that has been modified tocontain a nucleic acid segment that encodes a Chondroitin Synthaseprotein or fragment thereof. The recombinant vector may be furtherdefined as an expression vector comprising a promoter operatively linkedto said Chondroitin Synthase encoding nucleic acid segment.

A further preferred embodiment of the present invention is a host cellmade recombinant with a recombinant vector comprising a ChondroitinSynthase gene. The preferred recombinant host cell may be a prokaryoticcell. In another embodiment, the recombinant host cell is a eukaryoticcell. As used herein, the term “engineered” or “recombinant” cell isintended to refer to a cell into which a recombinant gene, such as agene encoding Chondroitin Synthase, has been introduced. Therefore,engineered cells are distinguishable from naturally occurring cellswhich do not contain a recombinantly introduced gene. Engineered cellsare thus cells having a gene or genes introduced through the hand ofman. Recombinantly introduced genes will either be in the form of a cDNAgene, a copy of a genomic gene, or will include genes positionedadjacent to a promoter not naturally associated with the particularintroduced gene.

Where one desires to use a host other than Pasteurella, as may be usedto produce recombinant chondroitin synthase, it may be advantageous toemploy a prokaryotic system such as E. coli, B. subtilis, Lactococcussp., or even eukaryotic systems such as yeast or the like. Of course,where this is undertaken it will generally be desirable to bring thechondroitin synthase gene under the control of sequences which arefunctional in the selected alternative host. The appropriate DNA controlsequences, as well as their construction and use, are generally wellknown in the art as discussed in more detail hereinbelow.

In preferred embodiments, the chondroitin synthase-encoding DNA segmentsfurther include DNA sequences, known in the art functionally as originsof replication or “replicons”, which allow replication of contiguoussequences by the particular host. Such origins allow the preparation ofextrachromosomally localized and replicating chimeric segments orplasmids, to which chondroitin synthase DNA sequences are ligated. Inmore preferred instances, the employed origin is one capable ofreplication in bacterial hosts suitable for biotechnology applications.However, for more versatility of cloned DNA segments, it may bedesirable to alternatively or even additionally employ originsrecognized by other host systems whose use is contemplated (such as in ashuttle vector).

The isolation and use of other replication origins such as the SV40,polyoma or bovine papilloma virus origins, which may be employed forcloning or expression in a number of higher organisms, are well known tothose of ordinary skill in the art. In certain embodiments, theinvention may thus be defined in terms of a recombinant transformationvector which includes the chondroitin synthase coding gene sequencetogether with an appropriate replication origin and under the control ofselected control regions.

Thus, it will be appreciated by those of skill in the art that othermeans may be used to obtain the Chondroitin Synthase gene or cDNA, inlight of the present disclosure. For example, polymerase chain reactionor RT-PCR produced DNA fragments may be obtained which contain fullcomplements of genes or cDNAs from a number of sources, including otherstrains of Pasteurella or from other bacterial sources, such aslibraries. Virtually any molecular cloning approach may be employed forthe generation of DNA fragments in accordance with the presentinvention. Thus, the only limitation generally on the particular methodemployed for DNA isolation is that the isolated nucleic acids shouldencode a biologically functional equivalent chondroitin synthase.

Once the DNA has been isolated it is ligated together with a selectedvector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovinepapilloma virus and retroviruses. However, it is believed thatparticular advantages will ultimately be realized where vectors capableof replication in both Lactococcus or Bacillus strains and E. coli or P.multocida are employed.

Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti or the pAT19 vector of Trieu-Cuot, et al., allow one to performclonal colony selection in an easily manipulated host such as E. coli,followed by subsequent transfer back into a food grade Lactococcus orBacillus strain for production of chondroitin. These are benign and wellstudied organisms used in the production of certain foods andbiotechnology products—otherwise known in the art as GRAS (GenerallyRegarded As Safe). GRAS organisms are advantageous in that one canaugment the Lactococcus or Bacillus strain's ability to synthesizechondroitin through gene dosaging (i.e., providing extra copies of theHA synthase gene by amplification) and/or the inclusion of additionalgenes to increase the availability of the chondroitin precursorsUDP-GlcUA and UDP-GalNAc. These precursors are made by the action ofUDP-glucose dehydrogenase and UDP-GlcNAc/UDP-GalNAc epimerase,respectively. The inherent ability of a bacterium to synthesizechondroitin can also be augmented through the formation of extra copies,or amplification, of the plasmid that carries the chondroitin synthasegene. This amplification can account for up to a 10-fold increase inplasmid copy number and, therefore, the Chondroitin Synthase gene copynumber.

Another procedure that would further augment Chondroitin Synthase genecopy number is the insertion of multiple copies of the gene into theplasmid. Another technique would include integrating the ChondroitinSynthase gene into chromosomal DNA. This extra amplification would beespecially feasible, since the Chondroitin Synthase gene size is small.In some scenarios, the chromosomal DNA-ligated vector is employed totransfect the host that is selected for clonal screening purposes suchas E. coli or Bacillus, through the use of a vector that is capable ofexpressing the inserted DNA in the chosen host. In certain instances,especially to confer stability, genes such as the chondroitin synthasegene, may be integrated into the chromosome in various positions in anoperative fashion. Unlike plasmids, integrated genes do not needselection pressure for maintenance of the recombinant gene.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1” is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:1. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table I, and also refers tocodons that encode biologically equivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ nucleic acid sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzyme activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, such as at least 90 to 95 percentsequence identity, or at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 18nucleotide (6 amino acid) positions, frequently over a window of atleast 24-48 nucleotide (8-16 amino acid) positions, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to the sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the comparisonwindow. The reference sequence may be a subset of a larger sequence.

Allowing for the degeneracy of the genetic code as well as conserved andsemi-conserved substitutions, sequences which have between about 40% andabout 99%; such as between about 60% and about 90%; or between about 80%and about 99% identity to the nucleotides of SEQ ID NO:1 or the aminoacids of SEQ ID NO:2 will be sequences which are “essentially as setforth” in SEQ ID NO:1 or 2. Sequences which are essentially the same asthose set forth in SEQ ID NO:1 may also be functionally defined assequences which are capable of hybridizing to a nucleic acid segmentcontaining the complement of SEQ ID NO:1 under “standard stringenthybridization conditions”, “moderately stringent hybridizationconditions”, “less stringent hybridization conditions”, or “lowstringency hybridization conditions”. Suitable “standard” or “lessstringent” hybridization conditions will be well known to those of skillin the art and are clearly set forth hereinbelow. In a preferredembodiment, standard stringent hybridization conditions or lessstringent hybridization conditions are utilized.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 60%, and moretypically with increasing homologies of at least 70%, 80% and 90%. Twoamino acid sequences are homologous if there is a partial or completeidentity between their sequences. For example, 60% homology means that60% of the amino acids are identical when the two sequences are alignedfor maximum matching. Gaps (in either of the two sequences beingmatched) are allowed in maximizing matching; gap lengths of 5 or lessare preferred with 2 or less being more preferred. Alternatively andpreferably, two protein sequences (or polypeptide sequences derived fromthem of at least 30 amino acids in length) are homologous, as this termis used herein, if they have an alignment score of at more than 5 (instandard deviation units) using the program ALIGN with the mutation datamatrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlasof Protein Sequence and Structure, pp. 101-110 (Volume 5, NationalBiomedical Research Foundation (1972)) and Supplement 2 to this volume,pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. The term“corresponds to” is used herein to mean that a polynucleotide sequenceis homologous (i.e., is identical, not strictly evolutionarily related)to all or a portion of a reference polynucleotide sequence, or that apolypeptide sequence is identical to a reference polypeptide sequence.In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA”.

The terms “standard stringent hybridization conditions,” “moderatelystringent conditions,” and “less stringent hybridization conditions” or“low stringency hybridization conditions” are used herein, describethose conditions under which substantially complementary nucleic acidsegments will form standard Watson-Crick base-pairing and thus“hybridize” to one another. A number of factors are known that determinethe specificity of binding or hybridization, such as pH; temperature;salt concentration; the presence of agents, such as formamide anddimethyl sulfoxide; the length of the segments that are hybridizing; andthe like. There are various protocols for standard hybridizationexperiments. Depending on the relative similarity of the target DNA andthe probe or query DNA, then the hybridization is performed understringent, moderate, or under low or less stringent conditions.

The hybridizing portion of the hybridizing nucleic acids is typically atleast about 14 nucleotides in length, and preferably between about 14and about 100 nucleotides in length. The hybridizing portion of thehybridizing nucleic acid is at least about 60%, e.g., at least about 80%or at least about 90%, identical to a portion or all of a nucleic acidsequence encoding a HAS or chondroitin or heparin synthase or itscomplement, such as SEQ ID NO:1 or the complement thereof. Hybridizationof the oligonucleotide probe to a nucleic acid sample typically isperformed under standard or stringent hybridization conditions. Nucleicacid duplex or hybrid stability is expressed as the melting temperatureor T_(m), which is the temperature at which a probe nucleic acidsequence dissociates from a target DNA. This melting temperature is usedto define the required stringency conditions. If sequences are to beidentified that are related and substantially identical to the probe,rather than identical; then it is useful to first establish the lowesttemperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g., SSC, SSPE, or HPB). Then,assuming that 1% mismatching results in a 1° C. decrease in the T_(m),the temperature of the final wash in the hybridization reaction isreduced accordingly (for example, if sequences having >95% identity withthe probe are sought, the final wash temperature is decreased by about5° C.). In practice, the change in T_(m) can be between about 0.5° C.and about 1.5° C. per 1% mismatch. Examples of standard stringenthybridization conditions include hybridizing at about 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, followed with washing in 0.2×SSC/0.1% SDSat room temperature or hybridizing in 1.8×HPB at about 30° C. to about45° C. followed by washing a 0.2-0.5×HPB at about 45° C. Moderatelystringent conditions include hybridizing as described above in 5×SSC\5×Denhardt's solution 1% SDS washing in 3×SSC at 42° C. The parameters ofsalt concentration and temperature can be varied to achieve the optimallevel of identity between the probe and the target nucleic acid.Additional guidance regarding such conditions is readily available inthe art, for example, by Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, (Cold Spring Harbor Press, N.Y.); and Ausubel et al.(eds.), 1995, Current Protocols in Molecular Biology, (John Wiley &Sons, N.Y.). Several examples of low stringency protocols include: (A)hybridizing in 5×SSC, 5× Denhardts reagent, 30% formamide at about 30°C. for about 20 hours followed by washing twice in 2×SSC, 0.1% SDS atabout 30° C. for about 15 min followed by 0.5×SSC, 0.1% SDS at about 30°C. for about 30 min (FEMS Microbiology Letters, 2000, vol. 193, p.99-103); (B) hybridizing in 5×SSC at about 45° C. overnight followed bywashing with 2×SSC, then by 0.7×SSC at about 55° C. (J. VirologicalMethods, 1990, vol. 30, p. 141-150); or (C) hybridizing in 1.8×HPB atabout 30° C. to about 45° C.; followed by washing in 1×HPB at 23° C.

As is well known in the art, most of the amino acids in a protein arepresent to form the “scaffolding” or general environment of the protein.The actual working parts responsible for the specific desired catalysisare usually a series of small domains or motifs. Thus a pair of enzymesthat possess the same or similar motifs would be expected to possess thesame or similar catalytic activity, thus be functionally equivalent.Utility for this hypothetical pair of enzymes may be consideredinterchangeable unless one member of the pair has a subset of distinct,useful properties. In a similar vein, certain non-critical motifs ordomains may be dissected from the original, naturally occurring proteinand function will not be affected; removal of non-critical residues doesnot perturb the important action of the remaining critical motifs ordomains. By analogy, with sufficient planning and knowledge, it shouldbe possible to translocate motifs or domains from one enzyme to anotherpolypeptide to confer the new enzyme with desirable characteristicsintrinsic to the domain or motif.

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:1. Nucleic acid sequences which are “complementary” arethose which are capable of base-pairing according to the standardWatson-Crick complementarity rules. As used herein, the term“complementary sequence” means a nucleic acid sequence which issubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that theiroverall length may vary considerably. It is therefore contemplated thata nucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Naturally, it will also be understood that this invention is not limitedto the particular nucleic acid and amino acid sequences of SEQ ID NOS:1and 2. Recombinant vectors and isolated DNA segments may thereforevariously include the Chondroitin Synthase coding regions themselves,coding regions bearing selected alterations or modifications in thebasic coding region, or they may encode larger polypeptides whichnevertheless include Chondroitin Synthase coding regions or may encodebiologically functional equivalent proteins or peptides which havevariant amino acids sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent Chondroitin Synthase proteins and peptides. Suchsequences may arise as a consequence of codon redundancy and functionalequivalency which are known to occur naturally within nucleic acidsequences and the proteins thus encoded. Alternatively, functionallyequivalent proteins or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein structuremay be engineered, based on considerations of the properties of theamino acids being exchanged. Changes designed by man may be introducedthrough the application of site-directed mutagenesis techniques, e.g.,to introduce improvements to the enzyme activity or to antigenicity ofthe Chondroitin Synthase protein or to test Chondroitin Synthase mutantsin order to examine chondroitin synthase activity at the molecularlevel.

Also, specific changes to the Chondroitin Synthase coding sequence canresult in the production of chondroitin having a modified sizedistribution or structural configuration. One of ordinary skill in theart would appreciate that the Chondroitin Synthase coding sequence canbe manipulated in a manner to produce an altered chondroitin synthasewhich in turn is capable of producing chondroitin having differingpolymer sizes and/or functional capabilities. For example, theChondroitin Synthase coding sequence may be altered in such a mannerthat the chondroitin synthase has an altered sugar substrate specificityso that the chondroitin synthase creates a new chondroitin-like polymerincorporating a different structure via the inclusion of a previouslyunincorporated sugar or sugar derivative. This newly incorporated sugarcould result in a modified chondroitin having different functionalproperties. As will be appreciated by one of ordinary skill in the artgiven the Chondroitin Synthase coding sequences, changes and/orsubstitutions can be made to the Chondroitin Synthase coding sequencesuch that these desired property and/or size modifications can beaccomplished.

Basic knowledge on the substrate binding sites (e.g., the UDP-GlcUA siteor UDP-GalNAc site or oligosaccharide acceptor site) of PmCS allows thetargeting of residues for mutation to change the catalytic properties ofthe site. The identity of important catalytic residues of PmCS as wellas PmHAS, a close homolog of PmCS, has recently been elucidated (Jing &DeAngelis, Glycobiology, 13: 661-671 (2003); and Jing & DeAngelis,Glycobiology, 10:883-889 (2000)). Appropriate changes at or near theseresidues would allow other UDP-sugars to bind instead of the authenticchondroitin sugar precursors; thus a new, modified polymer issynthesized. Polymer size changes will be caused by differences in thesynthase's catalytic efficiency or changes in the acceptor siteaffinity. Polymer size changes have been made in the vertebrate HAS,xlHAS1 (DG42), by mutating various residues (see parent application U.S.Ser. No. 11/042,530). As PmCS is a more malleable, robust enzyme thanthis latter enzyme, similar or superior versions of mutant PmCS whichsynthesize modified polymers are also possible.

The term “modified structure” as used herein denotes a chondroitinpolymer containing a sugar or derivative not normally found in thenaturally occurring chondroitin polypeptide. The term “modified sizedistribution” refers to the synthesis of chondroitin molecules of a sizedistribution not normally found with the native enzyme; the engineeredsize could be much smaller or larger than normal.

Various chondroitin products of differing size have application in theareas of drug delivery and the generation of an enzyme of alteredstructure can be combined with a chondroitin of differing size.Applications in angiogenesis, cancer treatment and wound healing arepotentially large if chondroitin polymers of about 10-20 monosaccharidescan be made in good quantities. Another particular application for smallchondroitin oligosaccharides is in the stabilization of recombinanthuman proteins used for medical purposes. A major problem with suchproteins is their clearance from the blood and a short biologicalhalf-life. One present solution to this problem is to couple a smallmolecule shield that prevents the protein from being cleared from thecirculation too rapidly. Very small molecular weight chondroitin is wellsuited for this role and would be nonimmunogenic and biocompatible.Larger molecular chondroitin attached to a drug or protein may be usedto target the reticuloendothelial cell system which has endocyticreceptors for chondroitin. Large polymers may be used in highconcentrations to make gels or viscous solutions with potential forjoint lubrications opthaltmic procedures, and cosmetics.

One of ordinary skill in the art given this disclosure would appreciatethat there are several ways in which the size distribution of thechondroitin polymer made by the chondroitin synthase could be regulatedto give different sizes. First, the kinetic control of product size canbe altered by decreasing temperature, decreasing time of enzyme actionand by decreasing the concentration of one or both sugar nucleotidesubstrates. Decreasing any or all of these variables will give loweramounts and smaller sizes of chondroitin product. The disadvantages ofthese approaches are that the yield of product will also be decreasedand it may be difficult to achieve reproducibility from day to day orbatch to batch.

Secondly, the alteration of the intrinsic ability of the enzyme tosynthesize a large chondroitin product. Changes to the protein can beengineered by recombinant DNA technology, including substitution,deletion and addition of specific amino acids (or even the introductionof prosthetic groups through metabolic processing). Such changes thatresult in an intrinsically slower enzyme could then allow morereproducible control of chondroitin size by kinetic means. The finalchondroitin size distribution is determined by certain characteristicsof the enzyme that rely on particular amino acids in the sequence.

Specific changes in any of these residues can produce a modifiedhyaluronan or chondroitin that produces a hyaluronan or chondroitinproduct having a modified size distribution. Engineered changes toseHAS, spHAS, pmHAS, cvHAS and pmCS that decrease the intrinsic size ofthe hyaluronan or chondroitin polymer that the enzyme can make beforethe hyaluronan or chondroitin is released will provide powerful means toproduce either a hyaluronan or chondroitin polymer product of smaller orpotentially larger size than the native enzyme.

Finally, larger molecular weight chondroitin made be degraded withspecific chondroitinases to make lower molecular weight chondroitin.This practice, however, is very difficult to achieve reproducibility andone must meticulously repurify the chondroitin to remove thechondroitinases and unwanted digestion products.

Structurally modified chondroitin is no different conceptually thanaltering the size distribution of the chondroitin product by changingparticular amino acids in the desired Chondroitin Synthase and/or moreparticularly, but not limiting thereto pmCS. Derivatives of UDP-GalNAc,in which the acetyl group is missing from the amide (UDP-GalN) orreplaced with another chemically useful group (for example, phenyl toproduce UDP-GalNPhe or propyl to produce UDP-GalNPro), is expected to beparticularly useful. The free amino group would be available forchemical reactions to derivatize chondroitin in the former case withGalN incorporation. In the latter case, GalNPhe or GalNPro, would makethe polymer more hydrophobic or prone to making emulsions. In addition,certain derivatives of GlcUA with extra chemical groups that alter thechemical and/or biological nature of the sugar should be useful. Thestrong substrate specificity may rely on a particular subset of aminoacids among the 10-20% that are conserved. Specific changes to one ormore of these residues create a functional chondroitin synthase thatinteracts less specifically with one or more of the substrates than thenative enzyme. This altered enzyme could then utilize alternate naturalor special sugar nucleotides to incorporate sugar derivatives designedto allow different chemistries to be employed for the followingpurposes: (i) covalently coupling specific drugs, proteins, or toxins tothe structurally modified chondroitin for general or targeted drugdelivery, radiological procedures, etc. (ii) covalently cross linkingthe hyaluronic acid itself or to other supports to achieve a gel, orother three dimensional biomaterial with stronger physical properties,(iii) covalently linking hyaluronic acid to a surface to create abiocompatible film or monolayer, and (iv) confer resistance todegradation.

In general, prokaryotes are used for the initial cloning of DNAsequences and construction of the vectors useful in the invention. It isbelieved that a suitable source may be bacterial cells, particularlythose derived from strains that can exist on a simple minimal media forease of purification. Bacteria with a single membrane but a thick cellwall, such as Staphylococci and Streptococci, are Gram-positive.Gram-negative bacteria such as E. coli contain two discrete membranesrather than one surrounding the cell. Gram-negative organisms tend tohave thinner cell walls. The single membrane of the Gram-positiveorganisms is analogous to the inner plasma membrane of Gram-negativebacteria. Additionally, many bacteria possess transport systems thathelp capsular polymers be secreted from the cell.

In general, plasmid vectors containing origins of replication andcontrol sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries an origin of replication, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. Anorigin of replication may be provided either by construction of thevector to include an exogenous origin or may be provided by the hostcell chromosomal replication mechanism. If the vector is integrated intothe host cell chromosome, the latter mechanism is often sufficient.

Many promoters have been discovered and utilized, and details concerningtheir nucleotide sequences have been published, enabling a skilledworker to ligate them functionally with plasmid vectors. Also for usewith the present invention one may utilize integration vectors.

In addition to prokaryotes, eukaryotic microbes such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other species (e.g., Pichia) are commonly available. Forexpression in Saccharomyces, the plasmid YRp7, for example, is commonlyused. This plasmid already contains the trp1 gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow without tryptophan, for example, ATCC No. 44076 or PEP4-1. Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Suitablepromoting sequences in yeast vectors include the promoters for thegalactose utilization genes, the 3-phosphoglycerate kinase or otherglycolytic enzymes, such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

In constructing suitable expression plasmids, the termination sequencesassociated with these genes are also ligated into the expression vector3′ of the sequence desired to be expressed to provide polyadenylation ofthe mRNA and termination. Other promoters, which have the additionaladvantage of transcription controlled by growth conditions are thepromoter region for alcohol dehydrogenase 2, cytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Any plasmidvector containing a yeast-compatible promoter, origin of replication andtermination sequences is suitable.

Chondroitin sulfate and dermatan sulfate are both derived from the samepolymer, i.e. D-glucuronic acid beta (1-3)D-N-acetyl galactosamine beta(1-4). Both chondroitin sulfate and dermatan sulfate can be sulfated atpositions 4 or 6 of N-acetyl galactosamine and position 2 of the uronicacid. Neither has been observed to be N-sulfated in nature. Thedifference between chondroitin sulfate and dermatan sulfate is theepimerisation of glucuronic acid to iduronic acid. There are problemshowever with the nomenclature and designation of a polysaccharide aseither chondroitin sulfate or dermatan sulfate. In particular, thefrequency with which iduronic acid must occur rather than glucuronicacid, for the chain to be called a dermatan sulfate chain, is open tointerpretation. Thus a chondroitin sulfate chain may have sequences ofdermatan sulfate interspersed therein and visa versa. One of ordinaryskill in the art would appreciate, however, that a polymer havingbetween 10% and 50% epimerisation of glucuronic acid to iduronic acidwould be suitably designated a dermatan sulfate polysaccharide.

A chondroitin polymer is produced by a chondroitin synthase and inparticular, but not limited thereto, the pmCS of the present invention.For example, the chondroitin polymer can be converted into a dermatanmolecule that may be an even more valuable product than chondroitinitself. The chondroitin polymer can be converted into dermatan either inthe purified form or in vivo (i.e. in the host itself). For example,Chang et al. have identified and detailed a reaction of Azotobactervinelandii poly-beta-D-mannuronic acid C-5-epimerase on syntheticD-glucuronans. A dermatan molecule can be made using the Azotobactervinelandii poly-beta-(1->4)-D-mannuronic acid C-5-epimerase to reactwith a chondroitin polymer made via a chondroitin synthase such as pmCS.(Chang et al. Action of Azotobacter vinelandii poly-beta-D-mannuronicAcid C-5-epimerase on Synthetic D-Glucuronans, Carbohydrate Research,Dec. 1, 2000; 329(4):913-22, which is expressly incorporated herein inits entirety by reference). U.S. Pat. No. 5,939,289 issued to Ertesvaget al., which is expressly incorporated herein by reference, alsodiscloses a C-5 epimerase which may be used to convert the chondroitinmolecule produced by the P. multocida chondroitin synthase into anunsulfated dermatan molecule. The C-5 epimerase is expected to work onthe chondroitin polymer as the Chang et al. paper describesepimerization of a variety of polysaccharides containing uronic acidsincluding oxidized starch and chitin.

Alternatively, instead of step-wise chondroitin synthesis followed byepimerization reaction, an in vivo combined method should be possible.This is very suitable in pmCS/Azotobacter epimerase case as thereactions are compatible and both genes are from Gram-negative bacteria.Both enzymes have been shown to function in E. coli. Placing both genesin one cell and allowing contact of chondroitin and the epimeraseresults in the desired product.

Further, an assay procedure for measuring the reactions catalyzed bypolyuronic acid C-5 epimerases can be used. (See e.g., Chang et al.Measurement of the Activity of Polyuronic Acid C-5 Epimerases, Anal.Biochem., Apr. 10, 1998; 258(1):59-62, which is expressly incorporatedherein in its entirety by reference) Action of C-5 epimerases invertsthe C-6 carboxyl group of polyuronic acids thus converting beta-linkedresidues into alpha-linked residues or vice versa. The above-identifiedassay takes advantage of the greater susceptibility of the acidhydrolysis of alpha-glycosidic linkages than beta-glycosidic linkages.Thus, acid treatment of experimental polymers (the product) results in acolor yield but the parent starting material does not result in asubstantial color yield. The method of this particular assay involvesthe partial acid hydrolysis of the polyuronic acid before and afterreaction with the C-5 epimerase. The greater or lesser amounts of uronicacid released (solubilized) before and after reaction of the C-5epimerase are a measure of the amount of alpha- or beta-glycosidiclinkages that are formed and a measure of the amount of catalysis by theenzyme. In this manner, the conversion chondroitin polymer to dermatancan be catalyzed and monitored for reaction and efficiency.

The chondroitin molecule made by the PmCS enzyme is an ideal polymericstarting material for the creation of a dermatan sulfate molecule.Certain mammalian epimerases only epimerize unsulfated polymermolecules. For example, the C-5 uronosyl epimerase, which is capable ofconverting a chondroitin molecule into a dermatan molecule, will onlyepimerize an unsulfated chondroitin molecule. Unsulfated chondroitinmolecules are not found in nature, and chondroitin sulfate must beeither desulfated or an unsulfated chondroitin molecule must berecombinantly produced. Since no chondroitin synthase has been knownprior to or since the discovery of the pmCS enzyme, one of ordinaryskill in the art would have to expend additional time, money, andcapital in order to convert sulfated chondroitin into unsulfated ordesulfated chondroitin. Once the chondroitin is unsulfated, themammalian epimerases can be used to convert the chondroitin moleculeinto a dermatan molecule. By utilizing a chondroitin synthase, such aspmCS, one of ordinary skill in the art is capable of producing anunsulfated chondroitin molecule which is an ideal starting material forepimerization by a mammalian epimerase. For one such mammalian epimeraseand methods of using same, see e.g. Malmstrom A., Biosynthesis ofDermatan Sulfate—Substrate Specificity of the C-5 Uronosyl Epimerase, J.Biol. Chem., Jan. 10, 1984; 259(1): 161-5, which is expresslyincorporated herein by reference in its entirety.

Utilizing enzymatic sulfation, the chondroitin polymer—turned—dermatanmolecule can be sulfated, thereby creating an even more valuable andflexible polymer for anticoagulation, device coatings, and/or otherbiomaterial. As pointed out in the Eklund et al. article entitledDermatan is a Better Substrate for 4-O-sulfation than Chondroitin:Implications in the Generation of 4-O-sulfated, L-iduronic-richGalactosaminoglycans, Arch. Biochem. Biophys., Nov. 15, 2000;383(2):171-7, which is expressly incorporated in its entirety herein byreference, dermatan is not only more easily enzymatically sulfated thanchondroitin, but sulfated dermatan is a more valuable, flexible anduseful product than chondroitin. Thus, utilizing a chondroitin synthasesuch as pmCS, one or ordinary skill in the art, given the presentdisclosure, would be able to produce natural and non-natural chondroitinas well as dermatan and dermatan sulfate. Other articles, which areexpressly incorporated herein in their entirety by reference, outlineother methodologies for enzymatically sulfating dermatan. Bhakta et al.Sulfation of N-acetylglucosamine by Chondroitin 6-Sulfotransferase 2(GST-5), J. Biol. Chem., Dec. 22, 2000; 275(51):40226-34; Ito et al.Purification and Characterization of N-acetylgalactosamine 4-sulfate6-O-Sulfotransferase from the Squid Cartilage, J. Biol. Chem., Nov. 3,2000; 275(44):34728-36.

In addition to enzymatic sulfation, the dermatan polymer can bechemically sulfated. One method for chemical sulfation is outlined inthe article by Garg et al. entitled Effect of Fully SulfatedGlycosaminoglycans on Pulmonary Artery Smooth Muscle Cell Proliferation,Arch. Biochem. Biophys., Nov. 15, 1999; 371(2): 228-33, which isexpressly incorporated herein in its entirety by reference. Typically,the polysaccharide in an anhydrous solvent is treated with sulfurtrioxide or chlorosulfonic acid. In any event, one of ordinary skill inthe art given the chondroitin synthase (PmCS) of the present inventionand the methodology for producing a chondroitin polymer from the PmCSenzyme would be capable of using the epimerization reaction to form adermatan molecule and then sulfating this dermatan molecule by knownenzymatic, or chemical sulfation techniques. Alternatively,unepimerimized chondroitin could be sulfated by any means as well.

Also, U.S. Pat. No. 4,990,601 issued to Skjak-Braek et al., which isexpressly incorporated herein by reference in its entirety, discloses achemical process using supercritical CO₂ which epimerizes uronic acid ina compound. Utilizing a chondroitin polypeptide using the pmCS of thepresent invention and the CO₂ epimerization method of Skjak-Braek etal., one of ordinary skill in the art can easily make unsulfateddermatan molecules.

An Example is provided hereinbelow. However, the present invention is tobe understood to not be limited in its application to the specificexperimentation, results and laboratory procedures. Rather, the Exampleis simply provided as one of various embodiments and is meant to beexemplary, not exhaustive.

EXAMPLE Materials and Methods

Materials and Pasteurella Strains—Unless otherwise noted, all chemicalswere from Sigma or Fisher, and all molecular biology reagents were fromPromega. The wild-type encapsulated Type F P. multocida strains, P-4679and P-3695, were obtained from Dr. Richard Rimler (USDA, Ames, Iowa).These strains were isolated from turkeys with fowl cholera. P-4679 had aslightly larger capsule than P-3695 as judged by light microscopy andIndia Ink staining.

Isolation of Capsule Biosynthesis Locus DNA—A lambda library of Sau3Apartially digested P-4679 DNA (4-9 kb average length insert) was madeusing the BamHI-cleaved Zap Express™ vector system (Stratagene). Theplaque lifts were screened by hybridization (5×SSC, 50° C.; 16 hrs) withthe digoxigenin-labeled probe using the manufacturer guidelines forcolorimetric development. E. coli XLI-Blue MRF′ was co-infected with thepurified, individual positive lambda clones and ExAssist helper phage toyield phagemids. The resulting phagemids were transfected into E. coliXLOLR cells to recover the plasmids. Sequence analysis of the plasmidsrevealed a novel open reading frame (ORF), i.e., PmCS (DeAngelis andPadgett-McCue, 200, J. Biol. Chem.). The central portion of both thePmCS and the PmHAS polypeptides (residues 430-530) is most homologous tobacterial glycosyltransferases from a wide variety of genera, includingStreptococcus, Vibrio, Neisseria and Staphylococcus, which formexopolysaccharides or the carbohydrate portions of lipopolysaccharides.Some of the most notable sequence similarities are the DGSTD and theD×DD motifs. Directly downstream of the pmCS gene a putative UDP-glucosedehydrogenase gene was identified. Therefore, the relative gene order[KfaA homolog—polysaccharide synthase gene—UDP-glucose dehydrogenasegene] in this portion of the Pasteurella Type F capsule operon is thesame as that found in Pasteurella Type A.

Construction and Expression of Recombinant P. multocida ChondroitinSynthase in Bacillus (FIG. 1). Construction of suitable vectorscontaining the desired coding and control sequences employ standardligation and transformation techniques. Isolated plasmids or DNAfragments are cleaved, tailored, and religated in the form desired toconstruct the plasmids required. The PmCS ORF (SEQ ID NO:1; encodingresidues 1-965 of SEQ ID NO:2) in the insert of one of the excisedlambda clones, pPmF4A (DeAngelis and Padgett-McCue, 200, J. Biol.Chem.), was amplified by polymerase chain reaction (PCR) with Taq or PfuDNA polymerase. The sense primer corresponded to the sequence at thededuced amino terminus of the ORF preceded by a Bacillus ribosomebinding sequence (RBS) and a unique restriction site compatible with theexpression plasmid. The antisense primer encoded the carboxyl terminusof the ORF followed by a stop codon and a second (i.e., different fromthe sense primer site) unique restriction site compatible with theexpression plasmids. The new restriction sites were used to facilitatethe cloning of the open reading frame downstream of the transcriptionalpromoter of the vector. The resulting PCR product encoding theRBS/synthase gene cassette was purified and concentrated usingGeneClean. The insert cassette was cleaved at the two flanking uniquerestriction sites (sites added with the two PCR primers above) andcloned into the two different plasmid-based expression vectors, pHCMC04and pHCMC05 (Bacillus Genetic Stock Center), cut with the restrictionenzymes to generate compatible ends suitable for ligation. Theseexpression vectors have been constructed allowing stable intracellularexpression of recombinant proteins in Bacillus subtilis strains (NguyenHG et al., Plasmid, 54:241-8 (2005)). These expression vectors are basedon the recently described Escherichia coli-B. subtilis shuttle vectorpMTLBS72 which replicates as theta circles. Two different controllablepromoters are used on pHCMC04 and pHCMC05: P(xylA) and P(spac),respectively, which respond to the addition of xylose and IPTG,respectively. The ligated products (FIG. 1; top panel) were transformedinto E. coli and plated on LB with ampicillin (100 μg/ml). In additionto the PmCS gene alone, a second series of constructs with the GalE gene(UDP-GlcNAc/GalNAc epimerase to help increase flux of UDP-GalNAcproduction; amplified by PCR from Bacillus subtilis genomic DNA withsuitable primers and Pfu polymerase) was also created (FIG. 1; bottompanel).

Colonies were analyzed by restriction digestion and DNA sequencing.Clones containing a plasmid with the desired ORF were transformed intoBacillus subtilis 168 (Bacillus Genetic Stock Center), theproduction/expression host, and maintained on LB media withchloramphenicol (5 μg/ml) at 30° C. Log phase cultures in definedsynthetic media (e.g., Spizzens) were induced withbeta-isopropylthiogalactoside (0.1-0.5 mM final) or xylose (0.1-0.5%final). The cells were removed by centrifugation, and the chondroitinwas purified by the CPC method (below) and tested.

Purification of Chondroitin—The anionic polymer in cultures of therecombinant Bacillus bacteria was purified by cetylpyridinium chloride(CPC) precipitation. Cells were grown in complete defined media (150 ml)with drug selection with mild shaking overnight at 37° C. Cells wereremoved by centrifugation (3,000×g, 10 min), and spent culture media washarvested. GAGs in the aqueous extract were precipitated by the additionof cetylpyridinium chloride (1% w/v final concentration). After standingfor 10 min, the precipitate was collected by high-speed centrifugationand redissolved in 2.5 M NaCl. The mixture was clarified by high-speedcentrifugation and the supernatant was precipitated with 3 vol ofethanol. The precipitate was washed with 70% ethanol, dried slightly,and resuspended in 2.5 M NaCl. The ethanol precipitation procedure wasrepeated, and the pellet was redissolved in water. Another round ofethanol precipitation (2 vol.) was performed. The final pellet wasdissolved in water.

Size Analysis and Enzymatic Degradation of Polymers (FIGS. 2 and 3). Gelelectrophoresis was used to analyze the size distribution of therecombinant polymers. Polymers were analyzed using 1 to 1.2% 1×TAEagarose gels (30 V, 5 h, Stains-All detection (Lee and Cowman, 1994)(FIG. 2). The testicular hyaluronidase from Sigma (St. Louis, Mo.) wasemployed to destroy and thus identify HA or chondroitin chains (note:this enzyme will digest both HA and chondroitin) (FIG. 3). Defined HAmolecular weight standards were from Hyalose L.L.C. (Oklahoma City,Okla.). Kilobase DNA standards were from Stratagene (La Jolla, Calif.).

Disaccharide Analysis—The FACE (fluorophore-assisted carbohydrateelectrophoresis) method was used to identify the sugar repeats of theGAGs produced (FIG. 4). In general, a sample (25 ¾g) was dissolved in100 μl 0.1 M ammonium acetate, pH 7.0 and 1 μl Chondroitinase ABC (16.6mU/μl; Sigma) was added followed by incubation at 37° C. (4 hrs toovernight). The sample was then lyophilized and re-dissolved in 40 μl2-aminoacridone HCl (12.5 mM dissolved in 85/15 dimethyl sulfoxide(DMSO)/acetic acid and incubated for 15 min in the dark at roomtemperature. Then 40 μl 1.25M sodium cyanoborohydride in water was addedfollowed by incubation at 37° C. The sample was lyophilized andre-dissolved before running the polyacrylamide gel in Tris borate buffer(Glycobiology, Vol. 13(1): 1G-3G, 2003). Approximately 0.5 micrograms ofsample/lane were run on the gel. Bands were detected by fluorescencewith ultraviolet light.

Detection of PmCS gene homologs. The pmCS gene DNA was used as ahybridization probe for detecting the E. coli K4 kfoC gene DNA (FIG. 5).Basically, a commercial Southern blot kit (Dig Hi-Prime, Roche) was usedto label restriction fragments containing pmCS with digoxigenin probe.This probe was used to analyze a Southern blot containing a PstI/EcoRIdigest of Type F Pasteurella multocida genomic DNA (a positive control;P lane), a PCR product of the kfoC gene (corresponding to product ofNinomiya et al., 2002; lane K), or Lambda HindIII standard (lane L). Thehybridization was carried out at 37° C. overnight in the manufacturer'sbuffer (Dig Easy Hyb). The blot was washed with 2×SSC, 0.1% SDS at 30°C. for 15 min twice, then for 30 min in 0.5×SSC, 0.1% SDS at 30° C.before using the manufacturer's Dig-antibody protocol for colorimetricdetection. The kfoC band is apparent (KfoC black arrow) as well as thenative Pasteurella gene (white arrow). No spurious hybridization signalswere seen from other irrelevant DNA species. Therefore, the knowledge ofthe pmCS sequence can be used to identify other chondroitin synthasecandidates for various uses (e.g., fermentation in vivo, production invitro) by known standard methodology.

Results/Discussion

Heterologous Expression of a Functional P. multocida ChondroitinSynthase—Previously, Western blot analysis using a monospecificantipeptide antibody was used to detect the production of pmCS¹⁻⁷⁰⁴ orpmHAS¹⁻⁷⁰³ polypeptide made in E. coli (see FIG. 2 of parent applicationU.S. Ser. No. 11/042,530). Both enzymes contain a sequence thatcorresponds exactly to the synthetic peptide used to generate theantibody. Extracts derived from E. coli cells containing the pmCS¹⁻⁷⁰⁴plasmid contained an immunoreactive band of the appropriate size (i.e.predicted to be 80 kDa), but this band was not present in samples fromcells with the vector alone control. Extracts derived from E. coli cellscontaining the pmCS¹⁻⁷⁰⁴ plasmid, but not samples from cells with thevector alone, synthesized chondroitin polymer in vitro when suppliedwith both UDP-GlcUA and UDP-GalNAc simultaneously (see Table 3 of U.S.Ser. No. 11/042,530).

The present invention demonstrates production of a chondroitin polymerby recombinant Bacillus possessing a pmCS gene (borne by a plasmid suchas in FIG. 1) in vivo. The high molecular weight polymer in the spentculture media was detected by agarose gel electrophoresis (0.8% gel runin 1×TAE system) (FIGS. 2 and 3). With StainsAll staining, HA and nativeP. multocida Type F polymer (=unsulfated chondroitin) or recombinantBacillus-derived polymer all stain blue. The recombinant polymer wassensitive to an enzyme treatment (FIG. 3) in a similar fashion toauthentic chondroitin. Any functional microbial chondroitin synthaseshould behave in a similar fashion to PmCS in a recombinant in vivosystem, especially if the two sequences are homologous or the catalystspossess similar reaction mechanisms. It is also expected that thelocation of the pmCS gene, either on a plasmid or integrated into thechromosome or present in a phage, is expected to be functional in therecombinant Gram-positive bacterial in vivo system as well.

The native and the recombinant chondroitin polymer are smaller than HAproduced by bacterial fermentation (FIG. 3), but still form very largechains of about 100 kDa to about 300 kDa. All other non-PmCS-derivedchondroitin reported in the literature has a size below 100 kDa. Suchhigher molecular weight polymers will have increased viscosity that ismore useful for certain medical devices and viscoelastic supplements.The polymer purified from recombinant cultures is a suitable startingmaterial for further processing (e.g., cleavage, cross-linking,sulfation, epimerization) into various therapeutics (e.g., biomaterials,nutraceuticals, or anticoagulants).

Compositional Analysis of Chondroitin Synthase-derived Polymers.Previous work by others has shown that the Type F capsule is removedfrom bacterial cells by treatment with chondroitin AC lyase. It wasfound that a fragment of the specific HA-binding protein, aggrecan, inthe HA-TEST assay (Pharmacia) did not cross-react with extracts of theType F polymer, but readily detected the HA in parallel extracts fromType A bacteria. Acid hydrolysis and monosaccharide analysis of the TypeF polymer showed that it contained the sugars galactosamine and GlcUA(Table 2 and FIG. 5 of U.S. Ser. No. 11/042,530). The NMR spectrum andother analyses of Type F polymer were also consistent with theunsulfated chondroitin structure (DeAngelis et al, 2002, Carb. ResearchVol 337:p. 1547-1522).

In general, it may be difficult to distinguish HA and unsulfatedchondroitin; these isomers only differ in the C4 position of theirhexosamine sugars. Experimental methods such as mass spectrometry alone,simple chemical assays, or many electrophoretic methods are notsufficient. However, FACE, a specialized electrophoretic method, is veryuseful for deciphering GAG disaccharide composition. Indeed, differentsugar isomers are readily distinguished, and this method is accepted asa ‘gold standard’ in glycobiology for many types of glycans (e.g.moieties from N- or O-glycoproteins) and monosaccharides (e.g. canseparate isobaric, isomeric sugars such as glucose, galactose, andmannose). In this invention, the FACE profile of the recombinantBacillus-derived chondroitin digest was indistinguishable from theauthentic unsulfated chondroitin (i.e. native Type F Pasteurella digest)(FIG. 4); the mixing experiments further demonstrated that the componentpeaks migrated identically. The chondroitin (both native andrecombinant) digests were easily distinguished from the HA digest, thusdemonstrating the power of FACE analysis.

Models of Production of GAGs with a Gram-negative GAG Synthase in aGram-positive Bacterial Host. The PmHAS protein was hypothesized tointeract with a putative polysaccharide transporter system or amembrane-bound partner via its carboxyl terminus, because deletion ofresidues 704 to 972 from the native-length enzyme resulted in theformation of a soluble enzyme (Jing and DeAngelis, 2000). However, nosubstantial membrane-associated or hydrophobic regions are predicted toreside in this sequence. As PmHAS and PmCS are highly homologous in thisregion, which is not essential for their glycosyltransferase activities,it was suggested that the carboxyl terminus contains domains or motifsrequired for interacting with the polysaccharide transport machinery ora membrane-bound partner in vivo.

Several hypotheses for production of a GAG by a Gram-negativePasteurella synthase in a Gram-positive host are possible, including: a)the synthase docks and uses Gram-positive polymer transport machinery,or b) the synthase can facilitate its own transport across a singleinner membrane. Empirically, the Gram-positive host, and morespecifically a Bacillus host, can support GAG production of aPasteurella GAG synthase; this finding is neither expected norpredicted, but clearly of utility to facilitate GAG manufacture in adefined, GRAS (generally regarded as safe), endotoxin-free fermentationsystem.

Some Gram-negative bacteria (e.g., E. coli) and some Gram-positivebacteria (B. subtilis) possess an UDP-GlcNAc/UDP-GalNAc epimerase(GalE). Therefore, the hexosamine precursor for chondroitin is availablefor polysaccharide biosynthesis without the need to gain an auxiliarymetabolic enzyme simultaneously, but adding an extra gene to alter thepool size of any required UDP-sugar is predicted to be favorable. Inconstructs using an auxiliary GalE gene behind the pmCS gene (FIG. 1,bottom) in Bacillus, the chondroitin yield is increased (FIG. 2); themost likely explanation is that with more epimerase protein (derivedfrom both chromosomal and man-made plasmid-derived genes, rather thanjust the original native state of one chromosomal endogenous GalE), theflux of GalNAc into the chondroitin polymer is increased.

Typically the UDP-glucose dehydrogenase, the enzyme that forms theUDP-GlcUA precursor, is found in Gram-negative bacteria only if themicrobe possesses a GlcUA-containing polymer or glycoconjugate. In bothType A and Type F P. multocida, the UDP-glucose dehydrogenase gene isdirectly downstream of the GAG synthase. Some Gram-positive bacteria (B.subtilis) also possess UDP-glucose dehydrogenase. But again boosting thelevel of any UDP-sugar by adding an extra gene to alter the pool size ofany UDP-sugar is predicted to be favorable. This enhancement was shownto be favorable with HA production in B. subtilis (Widner et al., Appl.Environ. Microbiol., 71: 3747-3752 (2005)). In addition, the eliminationof competing (e.g. sinks for UDP-sugars) or undesirable (e.g.,contaminant or detrimental molecule biosynthesis) pathways are alsoexpected to facilitate production of the target chondroitin polymers.

Thus, it should be apparent that there has been provided in accordancewith the present invention, methods of expressing a Gram-negativeglycosaminoglycan synthase gene in a Gram-positive host and productionof a glycosaminoglycan utilizing same, that fully satisfies theobjectives and advantages set forth above. Although the invention hasbeen described in conjunction with specific embodiments thereof, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications, and variations that fallwithin the spirit and broad scope of the appended claims.

1. A recombinant host cell, wherein the recombinant host cell is aGram-positive cell transformed, transduced or electroporated with arecombinant vector comprising a purified nucleic acid segment having acoding region encoding enzymatically active Pasteurella chondroitinsynthase, wherein the chondroitin synthase is a single protein that is adual-action transferase that catalyzes the polymerization of UDP-GlcUAand UDP-GalNAc to form chondroitin, wherein the recombinant host cellproduces chondroitin.
 2. The recombinant host cell of claim 1, whereinthe purified nucleic acid segment encodes the Pasteurella multocidachondroitin synthase of SEQ ID NO:2.
 3. The recombinant host cell ofclaim 1, wherein the purified nucleic acid segment comprises anucleotide sequence in accordance with SEQ ID NO:1.
 4. The recombinanthost cell of claim 1, wherein the purified nucleic acid segmenthybridizes to a complement of the nucleic acid segment of SEQ ID NO:1under hybridization conditions comprising 1.8× High Phosphate Buffer(HPB) at a temperature in a range of from about 30° C. to about 45° C.,followed by washing with 2×SSC/0.1% SDS at 30° C.
 5. The recombinanthost cell of claim 1, wherein the purified nucleic acid segment is60-99% identical to SEQ ID NO:1.
 6. The recombinant host cell of claim1, wherein the purified nucleic acid segment encodes an amino acidsequence of SEQ ID NO:2 with 0 to 20 conservative amino acidsubstitutions.
 7. The recombinant host cell of claim 1, wherein therecombinant host cell is selected from the group consisting of aBacillus cell, Staphylococcus cell, Peptococcus cell, Lactobacilluscell, Lactococcus cell, Actinomyces cell, and Streptomyces cell.
 8. Amethod for producing a chondroitin polymer in vivo, comprising the stepsof: providing a purified nucleic acid segment encoding an enzymaticallyactive Pasteurella chondroitin synthase, wherein the chondroitinsynthase is a single protein that is a dual-action transferase thatcatalyzes the polymerization of UDP-GlcUA and UDP-GalNAc to formchondroitin; placing the purified nucleic acid segment encoding anenzymatically active Pasteurella chondroitin synthase in a Gram-positivehost cell, thereby providing a recombinant Gram-positive host cellhaving a purified nucleic acid segment encoding an enzymatically activePasteurella chondroitin synthase therein; placing the recombinantGram-positive host cell in a medium suitable for the expression of achondroitin polymer; and extracting the chondroitin polymer.
 9. Themethod of claim 8 wherein, in the step of providing a purified nucleicacid segment encoding an enzymatically active Pasteurella chondroitinsynthase, the purified nucleic acid segment encodes the Pasteurellamultocida chondroitin synthase of SEQ ID NO:2.
 10. The method of claim 8wherein, in the step of providing a purified nucleic acid segmentencoding an enzymatically active Pasteurella chondroitin synthase, thepurified nucleic acid segment comprises a nucleotide sequence inaccordance with SEQ ID NO:1.
 11. The method of claim 8 wherein, in thestep of providing a purified nucleic acid segment encoding anenzymatically active Pasteurella chondroitin synthase, wherein thepurified nucleic acid segment hybridizes to a complement of the nucleicacid segment of SEQ ID NO:1 under hybridization conditions comprising1.8× High Phosphate Buffer (HPB) at a temperature in a range of fromabout 30° C. to about 45° C., followed by washing with 2×SSC/0.1% SDS at30° C.
 12. The method of claim 8 wherein, in the step of providing apurified nucleic acid segment encoding an enzymatically activePasteurella chondroitin synthase, the purified nucleic acid segment is60-99% identical to SEQ ID NO:1.
 13. The method of claim 8 wherein, inthe step of providing a purified nucleic acid segment encoding anenzymatically active Pasteurella chondroitin synthase, the purifiednucleic acid segment encodes an amino acid sequence of SEQ ID NO:2 with0 to 20 conservative amino acid substitutions.
 14. The method of claim 8wherein, in the step of placing the purified nucleic acid segment into aGram-positive host cell, the Gram-positive host cell is selected fromthe group consisting of a Bacillus cell, Staphylococcus cell,Peptococcus cell, Lactobacillus cell, Lactococcus cell, Actinomycescell, and Streptomyces cell.
 15. A method for producing a chondroitinpolymer, comprising the steps of: introducing a purified nucleic acidsegment having a coding region encoding enzymatically active Pasteurellachondroitin synthase into a Gram-positive host organism, wherein theGram-positive host organism contains nucleic acid segments encodingenzymes which produce UDP-GlcUA and UDP-GalNAc, and wherein thechondroitin synthase is a single protein that is a dual-actiontransferase that catalyzes the polymerization of UDP-GlcUA andUDP-GalNAc to form chondroitin; growing the Gram-positive host organismin a medium to secrete chondroitin polymer; and recovering the secretedchondroitin polymer.
 16. The method according to claim 15, wherein inthe step of recovering the chondroitin polymer, the chondroitin polymeris extracted from the medium or the cells or combinations thereof. 17.The method according to claim 16, further comprising the steps ofpurifying the extracted chondroitin polymer.
 18. The method according toclaim 15, further comprising the step of sulfating the chondroitinpolymer.
 19. The method according to claim 15, further comprising thestep of epimerizing the chondroitin polymer.
 20. The method of claim 15wherein, in the step of introducing a purified nucleic acid segmenthaving a coding region encoding enzymatically active Pasteurellachondroitin synthase into a Gram-positive host organism, the purifiednucleic acid segment encodes the Pasteurella multocida chondroitinsynthase of SEQ ID NO:2.
 21. The method of claim 15 wherein, in the stepof introducing a purified nucleic acid segment having a coding regionencoding enzymatically active Pasteurella chondroitin synthase into aGram-positive host organism, the purified nucleic acid segment comprisesa nucleotide sequence in accordance with SEQ ID NO:1.
 22. The method ofclaim 15 wherein, in the step of introducing a purified nucleic acidsegment having a coding region encoding enzymatically active Pasteurellachondroitin synthase into a Gram-positive host organism, wherein thepurified nucleic acid segment hybridizes to a complement of the nucleicacid segment of SEQ ID NO:1 under hybridization conditions comprising1.8× High Phosphate Buffer (HPB) at a temperature in a range of fromabout 30° C. to about 45° C., followed by washing with 2×SSC/0.1% SDS at30° C.
 23. The method of claim 15 wherein, in the step of introducing apurified nucleic acid segment having a coding region encodingenzymatically active Pasteurella chondroitin synthase into aGram-positive host organism, the purified nucleic acid segment is 60-99%identical to SEQ ID NO:1.
 24. The method of claim 15 wherein, in thestep of introducing a purified nucleic acid segment having a codingregion encoding enzymatically active Pasteurella chondroitin synthaseinto a Gram-positive host organism, the purified nucleic acid segmentencodes an amino acid sequence of SEQ ID NO:2 with 0 to 20 conservativeamino acid substitutions.
 25. The method of claim 15 wherein, in thestep of introducing a purified nucleic acid segment having a codingregion encoding enzymatically active Pasteurella chondroitin synthaseinto a Gram-positive host organism, the Gram-positive host organism isselected from the group consisting of a Bacillus cell, Staphylococcuscell, Peptococcus cell, Lactobacillus cell, Lactococcus cell,Actinomyces cell, and Streptomyces cell.